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Related Concept Videos

Two-dimensional Gel Electrophoresis01:22

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as  cells...
Electrophoresis: Overview01:20

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There...
Capillary Electrophoresis: Applications01:30

Capillary Electrophoresis: Applications

Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
Capillary Electrophoresis: Instrumentation01:20

Capillary Electrophoresis: Instrumentation

Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary for the separation by connecting to an anode (the positively charged electrode) and a cathode (the negatively charged electrode) located in buffer reservoirs at each end of the capillary tube. The system includes a sample vial, a fused silica capillary tube coated with polyimide for mechanical strength through which the sample components...
SDS-PAGE01:27

SDS-PAGE

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed  polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
Size-Exclusion Chromatography01:08

Size-Exclusion Chromatography

In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity,...

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Electrophoretic Separation of Proteins
08:17

Electrophoretic Separation of Proteins

Published on: June 12, 2008

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation.

Matthew R Richardson1, Sean Liu, Heather N Ringham

  • 1Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA.

Electrophoresis
|May 22, 2008
PubMed
Summary
This summary is machine-generated.

Solution isoelectric focusing (sIEF) effectively prefractionates complex bacterial lysates. This method significantly increases the number of detected protein spots in two-dimensional gel electrophoresis (2-DE) for improved proteome analysis.

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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
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Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method
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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
07:58

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Published on: September 19, 2018

Area of Science:

  • Proteomics
  • Biochemistry
  • Molecular Biology

Background:

  • Two-dimensional gel electrophoresis (2-DE) has limited utility for comprehensive proteome analysis due to the vast dynamic range and number of proteins.
  • Analyzing differential protein expression in complex samples like bacterial lysates is challenging with standard 2-DE, often detecting fewer than 2000 spots.

Purpose of the Study:

  • To enhance the analytical capacity of 2-DE for complex proteome studies, particularly for differential expression analysis.
  • To investigate the effectiveness of sample prefractionation using solution isoelectric focusing (sIEF) prior to 2-DE.

Main Methods:

  • Complex bacterial lysates were prefractionated using solution isoelectric focusing (sIEF) with the ZOOM IEF Fractionator.
  • Prefractionated samples were analyzed by parallel 2-DE using narrow-range immobilized pH gradient (IPG) strips.
  • The number of detected protein spots was quantified and compared to unfractionated samples.

Main Results:

  • Prefractionation via sIEF significantly increased the depth of proteome coverage resolved by 2-DE.
  • An average of 5525 protein spots were detected after sIEF prefractionation, a 3.5-fold increase compared to 1577 spots from unfractionated samples.
  • This approach facilitates the improved detection and analysis of low-abundance proteins.

Conclusions:

  • Solution isoelectric focusing (sIEF) is an effective prefractionation strategy to enhance the resolution and coverage of 2-DE.
  • This prefractionation method improves the ability to analyze complex proteomes and identify differentially expressed proteins.
  • sIEF coupled with 2-DE offers a powerful approach for in-depth proteomic investigations.